Inhibition of PfCLK3 directly affects RNA splicing and influence the splicing machinery.

Next generation antimalarials are required to be active across different Plasmodium species, offering cure and transmission blocking capability. Previously, we employed an extensive array of approaches (chemo-genetics, adaptive evolution followed by whole genome sequencing and mosquito membrane feeding assays) to validate the protein kinase PfCLK3 as an antimalarial target with these properties. Due to the close relationship of PfCLK3 to human kinases PRPF4B and CLK2 involved in key phosphorylation events that drive the assembly of the spliceosome, we hypothesise that the malaria kinase plays a role in splicing and RNA processing in Plasmodium parasites. However, no direct investigation has been carried out to confirm this postulation. Here, we present evidence that PfCLK3 inhibition by our tool compound TMCDC-135051 results in disruption of RNA-splicing. Following 1-hour exposure of 30-hours post invasion trophozoites to TCMDC-135051, RNA-sequencing revealed that RNA-splicing was extensively affected. In these experiments, multiple splice-junctions were mis-spliced in treated parasites compared to untreated controls. This effect was not seen in parasites expressing a mutant form of PfCLK3 (PfCLK3_G449P) insensitive to TCMDC-135051 – indicating the impact of the inhibitor on RNA-splicing in wild type parasites is via PfCLK3 inhibition. Importantly, analysis of the function of mis-spliced transcripts showed that the effected genes were involved in several essential parasite processes. In summary we established the role of PfCLK3 as a master regulator of spliceosome activity and RNA splicing in P. falciparum parasites and confirmed the mechanism of action of TMCDC-135051. This system can now be used to further investigate the mechanism of splicing in Apicomplexa and optimise CLK3 selective inhibitors. To investigate the effect of PfCLK3 inhibition on P falciparum 3D7 parasites, tightly synchronised wild type 3D7 and mutant parasites expressing mutant PfCLK3 (G449P) resistant to the PfCLK3 inhibitor were grown to a parasitaemia of 2-4% and 2% haematocrit upto 30-hours post invasion (30-hpi). Parasites were then treated with 1µM of TCMDC-135051, the selective PfCLK3 inhibitor for 1-hour under standard cultire conditions. The culture was spun down to pellet the cells and TRiZol was added as RNA preservative and stored at -80 until RNA purification. Using the purified RNA, we then prepare poly-A RNA librabry and proceed to sequencing using Illumina sequencing with upto 30-million paired-end reads. The RNA-sequencing data was used to investigate differential expression and differential splicing comparing resistant mutant and wild type parasites.